POS0290 NOVEL ANTI-MITOCHONDRIAL ANTIBODIES IDENTIFY PATIENTS WITH PROPENSITY TO THROMBOGENESIS IN SYSTEMIC LUPUS ERYTHEMATOSUS
Authors: M. Barguil Macedo, J. Chapa, T. Wang, A. Bengtsson, C. Lood
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Venous and arterial thrombotic events are leading causes of mortality in systemic lupus erythematosus (SLE), amounting to up to a quarter of deaths<sup>[1]</sup>. Other than traditional risk factors, SLE-specific factors, including chronic inflammation, have also been shown to contribute to thrombosis development. Nonetheless, the four times higher relative risk of thrombosis compared to healthy individuals is not fully accounted to the presence of conventional cardiovascular risk factors and anti-phospholipid antibodies<sup>[1]</sup>. As such, novel biomarkers of thrombosis are needed in early identification and preventative management of SLE patients. <h3>Objectives</h3> To characterize novel antibodies directed against mitochondrial constituents, and investigate their capacity to stratify patients based on thrombotic events. <h3>Methods</h3> Highly purified mitochondria were isolated from HepG2 cells (ATCC HB-8065TM) as previously described<sup>[2]</sup>. Briefly, cells were harvested and homogenized by a glass Douncer, followed by sequential centrifugation. Upon DNase-mediated removal of extracellular DNA, ultra-pure mitochondria (confirmed by flow cytometry, qPCR, and Western blot), labeled with MitoTracker, were incubated with sera (diluted 1:100) from SLE patients or healthy individuals. Binding of IgG to the mitochondrial outer membrane was detected using a secondary FITC-conjugated anti-human-IgG antibody, and analyzed by FACS. In some experiments, mitochondria were treated with trypsin (0.05%) prior to incubation with sera. Finally, reactivity towards mitochondrial protein lysate was confirmed using WB. Samples and clinical data from SLE patients (n=92) and healthy individuals (n=80) were provided from Division of Rheumatology, Lund University, Sweden. Statistical analyses were performed on SPSS 22.0. Mann-Whitney test was performed, with a p-value of 0.05 or below deemed as significant. Data protection and patient anonymization were ensured in accordance with regulation by Research Advisory Boards. <h3>Results</h3> Based on our novel flow cytometry assay, quantifying anti-mitochondrial antibodies (AMA) targeting the outer mitochondrial membrane, a large proportion (40.8%) of SLE patients were deemed positive for AMA using the 95<sup>th</sup> percentile of healthy controls as a cut-off. Presence of AMA was associated with severe SLE manifestations, including history of nephritis (OR = 3.3, p=0.02), anti-phospholipid syndrome (APS; OR = 5.7, p=0.02), and venous thromboembolism (OR = 6.7, p=0.008), the latter which was not seen in our cohort for isolated presence of anti-cardiolipin (aCL) antibodies (OR = 2.2, p=0.17), neither for lupus anticoagulant (OR 5.0, p=0.08) or anti-beta-2 glycoprotein 1 (OR 1.1, p=0.93). Pre-treatment of mitochondria with trypsin induced loss of binding of sera-derived antibodies (p&lt;0.01), suggesting that AMA were targeting protein components of mitochondria, and not phospholipids, as is the case of aCL. WB confirmed presence of reactivity towards mitochondrial protein antigens, in particular of 35 and 60 kDa, though reactivity towards other protein antigens, including 17 kDa ones, were also seen in some patients, with the latter being associated with APS (p=0.008). <h3>Conclusion</h3> SLE patients positive for novel AMAs targeting mitochondrial outer membrane proteins develop severe lupus manifestations, including venous thromboembolism. Future studies are warranted to further characterize the novel antibodies, as well as determine their prognostic value. <h3>References</h3> [1]Bello N, Meyers KJ, Workman J, et al. Systematic Literature Review and Meta-analysis of Venous Thromboembolism Events in Systemic Lupus Erythematosus. Rheumatol Ther. 2022 Dec 6. doi: 10.1007/s40744-022-00513-1. [2]Moore RE, Wang T, Duvvuri B, et al. Anti-mitochondrial antibodies predict erosive disease development in rheumatoid arthritis. Arthritis Rheumatol. 2022 Dec 29. doi: 10.1002/art.42428. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None declared.